STIM1 This Project: This web page originated as an assignment in Emory University's Biology 142 lab course. Students were assigned proteins of interest and asked to research what is known about the protein and to examine whether the newly sequenced whale shark genome had evidence of an orthologous protein.
Background Information: STIM 1, which stands for Stromal Interaction Molecule is a protein that encodes for a type 1 trans-membrane protein that mediates the passage of calcium ions through certain channels.(2) This gene is heavily coded for in the endoplasmic reticulum and is located in a very important region that is thought to be essential in tumor suppression.(1) Therefore, STIM1 is indeed an oncogene and Mutations or alterations in this area have been contributed to many diseases such as Bechwith-Wiedermann syndrome, Adrenocorticol carcinomas, ovarian and breast cancer, and many other harmful disorders. STIM1 is one of the main underlying components of the SOCE system(Store-Operated Calcium Entry), which regulates the influx of calcium ions in the endoplasmic reticulum. In 2005, a study done by Roos et al demonstrated that the destruction of STIM1 in human kidney cell led to a significant reduction in calcium levels, while an over expression of the gene resulted in increased calcium levels.(3) Over expression of this gene has been found to promote metastasis in Colorectal cancer cells both in vivo and in vitro. In fact, patients with positive STIM1 were found to have a more negative prognosis than patients that had a negative STIM1 according to Zhang et al. Figure 1
Figure 1: Immune system activation with STIM1:The figure above illustrates the pathway of the STIM1 gene function as it relates to the calcium influx pathway in the Endoplasmic Reticulum. STIM1 is a very essential gene in the immune response. Store-operated Calcium Entry (SOCE) is the primary mechanism of many immune cells in the immune response, as the depletion of calcium ions in the endoplasmic reticulum directs an influx of calcium ions. There is a specific complex that mediates this influx called the CRAC channels that are encoded by ORAI 1, 2, and 3 genes. This is where STIM1 comes in. Both STIM1 and 2 genes encode for proteins that act as sensors to activate the ORAI channels by sensing calcium depletion in the Endoplasmic Reticulum.
Methods/Approach The amino acid sequence of the gene was found using Ensembl databases by using the gene’s Ensemble ID (ENSP00000300737). This was done by entering the Ensemble ID, and then selecting ‘human’ as the species to search against. A transcript associated with the specific protein ID was chosen. BLASTp was then performed on the amino acid sequence. Once a good match was found from the BLAST results, similar sequences in other species were found using the NCBI Blast program (http://blast.ncbi.nlm.nih.gov/Blast.cgi). Homologues were found in particular species by selecting the ‘protein blast’ option and running the search. The FASTA format of the sequence was inserted into the ‘query sequence’ box, ‘blastp’ was the selected tab, and the sequence was “BLASTed.” The whale shark database was imported from the Galaxy server (whaleshark.georgiaaquarium.org). Imported data appeared as a green numbered item. The human sequence of the gene was entered as the query sequence by selecting ‘get data’ and then ‘upload data.’ The FASTA format of the sequence was then entered into the text box and then executed. Once the whale shark database and the query sequence were both uploaded to the history, a BLAST search was performed by clicking ‘NCBI Blast+.’ The pasted sequence was chosen as the query and the ‘FASTA file from history’ was chosen as the subject database. E-value and length of alignment were used to determine if any of the results were significant hits in the whale shark database. Once a significant BLAST hit was discovered, its sequence was retrieved. ‘Whale-shark.predicted-genes’ database was selected and the sequence ID of the predicted gene was entered. To align sequences, Clustal algorithm was used. The sequence alignment was then used to create the phylogenetic tree.
Protein Sequencing:
Figure 2:
Figure 2 Illustrates the four super families (Sam_Stim1, PRK13461, Mod_r, Bar, and SOAR) that we found using blast. SOAR is located in the endoplasmic reticulum and is classified as a metazoan transmembrane protein. Its function includes acting as a sensor for ER calcium ion levels and activates store-operated Ca2+ influx channels. STIM1 was found to accumulate near the plasma membrane and contact Orai1. STIM1 also has been found to play a role in sensing oxidative and temperature- variation stress and in mediating calcium signaling in response to stress. Modifier of rudimentary (Modr) proteins, represents a conserved region near 150 residues long within a number of eukaryotic proteins. Members of this family of protein include the VPS37 subunit of the endosomal sorting complex ESCRT-1. It functions in recruiting transport machinery for protein sorting at the multivesicular body. The Bin/Amphiphysin/Rvs (BAR) domain, which is a dimerization unit that binds membranes and detects membrane curvature. BAR is found in many different proteins with many different functions. The functions include organelle biogenesis, membrane trafficking or remodeling, and cell division/ migration. They may also be involved in protein-protein interactions. The Sterile Alpha Motif (SAM) domain is a unit consisting of about 70 amino acids. It is found in the Fungi/ Metazoa group and in a limited amount of bacteria. Proteins with this domain have a variety of domain structures and have various intracellular localization, involving the nucleus, cytoplasm, and membranes. Some functions include “interacting with proteins, RNA’s and membrane lipids, contain site of phosphorylation and kinase docking site, and play a role in protein homo and hetero dimerization/ oligomerization in processes ranging from signal transduction to regulation of transcription” (NCBI) (4).
Phylogeny:
Figure 3
Figure 3 illustrates the comparison of the closest hit we found from the Whale Shark data base, g37094, to other species when we cross referenced this gene from the galaxy database
Figure 4 illustrates the orthologs that we found matched with the human gene. As you can observe, all of the species in this table have some form of the gene present looking at the e-value and query coverage
Figure 5
Figure 5 shows the different gene sequences in the galaxy database that aligned somewhat with the human STIM1 protein sequence
Conclusion The STIM1 protein is present in the whaleshark. This suggests the protein did not prune away before the whaleshark was introduced in the phylogenetic tree. This means that the whaleshark has a protein that supports tumor suppression. Mutations and alterations of the protein would likely result in problems similar to that of humans.
References:
1)Bocchini, Carol A. "Stromal Interaction Molecule; STIM1." Www.OMIM.org. N.p., May-June 2001. Web.
2)"Stromal Interaction Molecule 1." Www.genecards.org. Weizmann Institute of Science, n.d. Web.
3)Zhang, Z. "STIM1, a Direct Target of MicroRNA-185, Promotes Tumor Metastasis and Is Associated with Poor Prognosis in Colorectal Cancer." Nature.com. Nature Publishing Group, n.d. Web. 31 Mar. 2015.
This Project:
This web page originated as an assignment in Emory University's Biology 142 lab course. Students were assigned proteins of interest and asked to research what is known about the protein and to examine whether the newly sequenced whale shark genome had evidence of an orthologous protein.
Background Information: STIM 1, which stands for Stromal Interaction Molecule is a protein that encodes for a type 1 trans-membrane protein that mediates the passage of calcium ions through certain channels.(2) This gene is heavily coded for in the endoplasmic reticulum and is located in a very important region that is thought to be essential in tumor suppression.(1) Therefore, STIM1 is indeed an oncogene and Mutations or alterations in this area have been contributed to many diseases such as Bechwith-Wiedermann syndrome, Adrenocorticol carcinomas, ovarian and breast cancer, and many other harmful disorders. STIM1 is one of the main underlying components of the SOCE system(Store-Operated Calcium Entry), which regulates the influx of calcium ions in the endoplasmic reticulum. In 2005, a study done by Roos et al demonstrated that the destruction of STIM1 in human kidney cell led to a significant reduction in calcium levels, while an over expression of the gene resulted in increased calcium levels.(3) Over expression of this gene has been found to promote metastasis in Colorectal cancer cells both in vivo and in vitro. In fact, patients with positive STIM1 were found to have a more negative prognosis than patients that had a negative STIM1 according to Zhang et al.
Figure 1
Figure 1: Immune system activation with STIM1:The figure above illustrates the pathway of the STIM1 gene function as it relates to the calcium influx pathway in the Endoplasmic Reticulum. STIM1 is a very essential gene in the immune response. Store-operated Calcium Entry (SOCE) is the primary mechanism of many immune cells in the immune response, as the depletion of calcium ions in the endoplasmic reticulum directs an influx of calcium ions. There is a specific complex that mediates this influx called the CRAC channels that are encoded by ORAI 1, 2, and 3 genes. This is where STIM1 comes in. Both STIM1 and 2 genes encode for proteins that act as sensors to activate the ORAI channels by sensing calcium depletion in the Endoplasmic Reticulum.
Methods/Approach
The amino acid sequence of the gene was found using Ensembl databases by using the gene’s Ensemble ID (ENSP00000300737). This was done by entering the Ensemble ID, and then selecting ‘human’ as the species to search against. A transcript associated with the specific protein ID was chosen. BLASTp was then performed on the amino acid sequence. Once a good match was found from the BLAST results, similar sequences in other species were found using the NCBI Blast program (http://blast.ncbi.nlm.nih.gov/Blast.cgi). Homologues were found in particular species by selecting the ‘protein blast’ option and running the search. The FASTA format of the sequence was inserted into the ‘query sequence’ box, ‘blastp’ was the selected tab, and the sequence was “BLASTed.” The whale shark database was imported from the Galaxy server (whaleshark.georgiaaquarium.org). Imported data appeared as a green numbered item. The human sequence of the gene was entered as the query sequence by selecting ‘get data’ and then ‘upload data.’ The FASTA format of the sequence was then entered into the text box and then executed. Once the whale shark database and the query sequence were both uploaded to the history, a BLAST search was performed by clicking ‘NCBI Blast+.’ The pasted sequence was chosen as the query and the ‘FASTA file from history’ was chosen as the subject database. E-value and length of alignment were used to determine if any of the results were significant hits in the whale shark database. Once a significant BLAST hit was discovered, its sequence was retrieved. ‘Whale-shark.predicted-genes’ database was selected and the sequence ID of the predicted gene was entered. To align sequences, Clustal algorithm was used. The sequence alignment was then used to create the phylogenetic tree.
Protein Sequencing:
Figure 2:
Figure 2 Illustrates the four super families (Sam_Stim1, PRK13461, Mod_r, Bar, and SOAR) that we found using blast. SOAR is located in the endoplasmic reticulum and is classified as a metazoan transmembrane protein. Its function includes acting as a sensor for ER calcium ion levels and activates store-operated Ca2+ influx channels. STIM1 was found to accumulate near the plasma membrane and contact Orai1. STIM1 also has been found to play a role in sensing oxidative and temperature- variation stress and in mediating calcium signaling in response to stress. Modifier of rudimentary (Modr) proteins, represents a conserved region near 150 residues long within a number of eukaryotic proteins. Members of this family of protein include the VPS37 subunit of the endosomal sorting complex ESCRT-1. It functions in recruiting transport machinery for protein sorting at the multivesicular body. The Bin/Amphiphysin/Rvs (BAR) domain, which is a dimerization unit that binds membranes and detects membrane curvature. BAR is found in many different proteins with many different functions. The functions include organelle biogenesis, membrane trafficking or remodeling, and cell division/ migration. They may also be involved in protein-protein interactions. The Sterile Alpha Motif (SAM) domain is a unit consisting of about 70 amino acids. It is found in the Fungi/ Metazoa group and in a limited amount of bacteria. Proteins with this domain have a variety of domain structures and have various intracellular localization, involving the nucleus, cytoplasm, and membranes. Some functions include “interacting with proteins, RNA’s and membrane lipids, contain site of phosphorylation and kinase docking site, and play a role in protein homo and hetero dimerization/ oligomerization in processes ranging from signal transduction to regulation of transcription” (NCBI) (4).
Phylogeny:
Figure 3
Figure 3 illustrates the comparison of the closest hit we found from the Whale Shark data base, g37094, to other species when we cross referenced this gene from the galaxy database
Figure 4
Figure 5
Figure 5 shows the different gene sequences in the galaxy database that aligned somewhat with the human STIM1 protein sequenceConclusion
The STIM1 protein is present in the whaleshark. This suggests the protein did not prune away before the whaleshark was introduced in the phylogenetic tree. This means that the whaleshark has a protein that supports tumor suppression. Mutations and alterations of the protein would likely result in problems similar to that of humans.
References:
1)Bocchini, Carol A. "Stromal Interaction Molecule; STIM1." Www.OMIM.org. N.p., May-June 2001. Web.
2)"Stromal Interaction Molecule 1." Www.genecards.org. Weizmann Institute of Science, n.d. Web.
3)Zhang, Z. "STIM1, a Direct Target of MicroRNA-185, Promotes Tumor Metastasis and Is Associated with Poor Prognosis in Colorectal Cancer." Nature.com. Nature Publishing Group, n.d. Web. 31 Mar. 2015.
4) "Basic Local Alignment Search Tool." BLAST:. N.p., n.d. Web. 14 Apr. 2015.
5) "Ensembl Genome Browser." Ensembl Genome Browser. N.p., n.d. Web. 14 Apr. 2015.
6) Galaxy/ Whale Shark. Coca Cola/ Georgia Aquarium, n.d. Web. 14 Apr. 2015. <http://whaleshark.georgiaaquarium.org/>.